REVIEW: Multiparametric Determination of Genes and Their Point Mutations
for Identification of Beta-Lactamases

2Institute of Technical Biochemistry, University of
Stuttgart, Stuttgart, Germany

* To whom correspondence should be addressed.

Received February 1, 2010; Revision received April 19, 2010
More than half of all currently used antibiotics belong to the
beta-lactam group, but their clinical effectiveness is severely limited
by antibiotic resistance of microorganisms that are the causative
agents of infectious diseases. Several mechanisms for the resistance of
Enterobacteriaceae have been established, but the main one is
the enzymatic hydrolysis of the antibiotic by specific enzymes called
beta-lactamases. Beta-lactamases represent a large group of genetically
and functionally different enzymes of which extended-spectrum
beta-lactamases (ESBLs) pose the greatest threat. Due to the plasmid
localization of the encoded genes, the distribution of these enzymes
among the pathogens increases every year. Among ESBLs the most
widespread and clinically relevant are class A ESBLs of TEM, SHV, and
CTX-M types. TEM and SHV type ESBLs are derived from penicillinases
TEM-1, TEM-2, and SHV-1 and are characterized by several single amino
acid substitutions. The extended spectrum of substrate specificity for
CTX-M beta-lactamases is also associated with the emergence of single
mutations in the coding genes. The present review describes various
molecular-biological methods used to identify determinants of
antibiotic resistance. Particular attention is given to the method of
hybridization analysis on microarrays, which allows simultaneous
multiparametric determination of many genes and point mutations in
them. A separate chapter deals with the use of hybridization analysis
on microarrays for genotyping of the major clinically significant
ESBLs. Specificity of mutation detection by means of hybridization
analysis with different detection techniques is compared.
KEY WORDS: antibiotic resistance, beta-lactamases, gene
determination, single nucleotide polymorphism, hybridization analysis,
microarrays

Microorganisms of the Enterobacteriaceae family are among the
most widespread pathogens of infectious diseases including hospital
infections. Beta-lactam antibiotics, which at the present time make up
more than half of antibiotics used in the world, are applied as
antibacterial drugs for treatment of these diseases. However, more and
more frequent cases of clinical inefficiency of drug therapy by this
group of antibiotics appear due to development of drug resistance among
the microorganisms. This biological phenomenon is called
“antibiotic resistance”. The problem of bacterial
resistance to antimicrobial preparations emerged practically
simultaneously with the beginning of use of antibiotics in the 1940s.
Already a year after the beginning of the use of penicillin, an enzyme
destroying this antibiotic, penicillinase, was detected in
Staphylococcus aureus. Data concerning microorganisms resistant
to certain antibiotic groups appeared in the 1970s, while already at
the end of that century microbial strains resistant to all known
antibiotics were described. Thus, the study of problems associated with
resistance to antibiotics as well as searching for ways of addressing
these problems became prominent for the world.

The main mechanism of emergence of resistance of the
Enterobacteriaceae family to beta-lactam antibiotics is the
appearance in their genes of chance mutations able to change the
spectrum of bacterial enzyme activities. Bacterial enzymes able to
cleave beta-lactam antibiotics are known as beta-lactamases.
Beta-lactamases comprise a superfamily of genetically and functionally
different enzymes joined by the ability to destroy the beta-lactam
ring, which results in the loss by the antibiotic of its antimicrobial
activity. Gram-negative bacteria of the Enterobacteriaceae
family that are resistant to the third generation of cephalosporins are
now the main problem in clinical practice. They produce the so-called
extended spectrum beta-lactamases (ESBL) [1]. The
first ESBLs, derivatives of the broad spectrum beta-lactamases, were
discovered in the mid 1980s. Now over 300 such enzymes are described,
and the number is constantly increasing.

ESBLs have been detected in all members of the Enterobacteriaceae
family as well as in Pseudomonas aeruginosa and Acinetobacter
baumannii. In most cases the ESBL genes are localized on plasmids,
and this is the reason for extremely rapid spreading of resistant
pathogens all over the world [2, 3]. This makes difficult correct diagnosis and choice
of an adequate method of treatment, which increases the number of cases
of inefficient therapy, lengthening and increasing the cost of therapy
[4, 5]. Thus, the need for
efficient laboratory diagnosis of ESBL production by Gram-negative
microorganisms is obvious. The difficulty of detection of
beta-lactamase in routine practice should be considered as an important
feature of these enzymes. Standard clinical methods of determination of
the nature of pathogens causing infectious diseases and revealing their
resistance to antibiotics are based on phenotypic characteristics of
microbial pathogens. At best, these methods of ESBL detection make it
possible to determine that the enzyme is present, but they cannot give
information about any particular enzyme. Besides, it is difficult to
use these methods in analysis of simultaneous multiple resistance to
several groups of antibiotics, whereas the number of such cases is
increasing.

The geographical diffusion of β-lactamases and emergence of
multiple resistances make necessary simultaneous determination of
several genes and their mutations, i.e. multiparametric analysis.
Molecular-biological methods for gene analysis are useful for solution
of this kind of problem. Investigations in chemistry and biochemistry
of nucleic acids during last decades not only resulted in revolutionary
advances in biology, but they exerted a pronounced effect on
development of new methods in biotechnology and gene engineering,
including technologies that can be applied for multiparametric analysis
of gene structure and detection of point mutations at the molecular
level. Recently several reviews have been published that deal with the
resistance of microorganisms to beta-lactam antibiotics and the
spreading and properties of beta-lactamase family enzymes [6-9]. However, there are no
reviews concerning molecular-biological methods for detection of
beta-lactamases enabling simultaneous detection of several antibiotic
resistance determinants. The goal of this review was to generalize all
available data in this field. Separate sections deal with methods for
multiparametric hybridization analysis for detection of several
antibiotic resistance determinants and ESBL identification.

ANTIBIOTIC RESISTANCE CAUSED BY BETA-LACTAMASES

The term “antibiotic resistance” characterizes resistance of
bacterial pathogens of infectious diseases to antibiotics and is a
particular case of a more common event of “antimicrobial
resistance”, resistance of pathogens of different nature
infectious (bacteria, viruses, protozoa) to drug preparations. One of
the most widely used classes of antibiotics is beta-lactam antibiotics
(beta-lactams). Beta-lactams are polar hydrophilic compounds
penetrating into bacterial cells through porin channels of the outer
membrane [10]. Beta-lactams are divided into
several groups based on their chemical structures having a beta-lactam
ring as the component in common (Table 1).
Cephalosporins are most often used for treatment of infections caused
by Gram-positive and Gram-negative bacteria. Antibiotics of the
penicillin group are most often used for treatment of infections caused
by Gram-positive microbial pathogens. The use of carbapenems as wide
spectrum antibiotics has recently increased.

Table 1. Structure of the main groups of
beta-lactam antibiotics (the beta-lactam ring is designated by the
dotted line)

The action mechanism of beta-lactam includes binding to
penicillin-binding proteins (PBP) – trans- and carboxypeptidases
involved in formation of peptidoglycan chains of the inner bacterial
membrane. The interaction of PBP with beta-lactam antibiotics results
in disturbance of peptidoglycan synthesis, cessation of cell division,
and cell death. The binding of the antibiotic to PBP is based on the
affinity of the beta-lactam structure to that of the PBP active site.
Owing to this, the presence of a beta-lactam ring is necessary for the
antibacterial activity of the antibiotic. Upon the interaction of the
beta-lactam with PBP, an acyl-enzyme complex is formed and the
C–N bond in the four-membered cycle of the beta-lactam ring is
broken.

Point mutations emerged during evolution of some PBP, which resulted in
appearance of beta-lactamases able to hydrolyze the lactam ring of
antibiotics. Comparative analysis of all PBP and beta-lactamase
sequences supports a hypothesis concerning the existence of a common
precursor of these proteins [11].

The following mechanisms of emergence of bacterial resistance to
beta-lactam antibiotics have been revealed: (i) synthesis of
beta-lactamases destroying these antibiotics [11-13]; (ii) lowering the
bacterial outer membrane permeability due to the loss of or decrease in
porin expression [14]; (iii) change in PBP
structure [15]; (iv) active antibiotic extrusion
from the microbial cell (efflux system) [16, 17]. Beta-lactamase synthesis is considered as the
main mechanism providing resistance of clinically important strains of
Gram-negative bacteria to beta-lactam antibiotics [6, 18, 19].
Genetic mutations resulting in replacement of just a few amino acids in
a protein sequence alter the enzyme structure resulting in significant
broadening of the antibiotic spectrum subjected to hydrolysis [20, 21]. Mutations can emerge
rather quickly; there are cases when microorganisms became resistant to
antibiotics during the course of treatment.

Resistance can be inborn or acquired. The real natural resistance is
characterized by the absence from microorganisms of a target for
revealing antibiotic activity. Some microorganisms are able to produce
chromosome-encoded beta-lactamases, for example, Klebsiella
pneumoniae produces beta-lactamase SHV-1 and Enterobacter
cloacae, Enterobacter aerogenes, Citrobacter
freundii, Serrata spp., and Pseudomonas
aeruginosa produce C class beta-lactamases. The ability of
individual bacterial strains to retain viability at antibiotic
concentrations inhibiting the bulk of the microbial population is
considered as acquired or secondary resistance. Situations are possible
when most of the population exhibits the acquired resistance. Formation
of acquired resistance is always due to acquiring of new genetic
information or to alteration of the native gene expression level. Most
often secondary resistance determinants are acquired together with
mobile genetic elements. The resistance of Enterobacteriaceae
family members to beta-lactam antibiotics is acquired resistance. The
ESBL genes are transferred by different mobile genetic elements like
plasmids, transposons, IS-elements, class 1 integrons, and the ISCR1
element containing integrons [22, 23]. The variety of mechanisms of genetic transfer
contributes to rapid spreading of these genes. From the point of view
of diagnosis, just detection of emergence and transfer of acquired
resistance comprises the main problem because it is impossible to
forecast its presence in the infectious pathogen.

Classification of
Beta-Lactamases

Over 500 different beta-lactamases have been
described, and this number is rapidly increasing each year. This enzyme
family itself can be called a superfamily because it joins several huge
groups or subfamilies differing in enzymatic properties. They are
united on the basis of their ability to hydrolyze beta-lactam
antibiotics, whereas differences include enzyme origin, amino acid
sequences, spectrum of substrate specificity, catalytic parameters, and
sensitivity to inhibitors. Numerous attempts have been made to classify
these enzymes. The first methods of classification were based on
comparison of their functional activity, namely on the ability of the
enzyme to cleave different classes of beta-lactam compounds
(penicillins, cephalosporins, monobactams, carbapenems), sensitivity to
inhibitors, and differences in biochemical parameters [24-26]. As a result, a scheme of
beta-lactamase classification by several functional groups was designed
[27]. A different beta-lactamase classification,
molecular classification based on a collection of common structural
features, was proposed as well [28]. Four
molecular classes are distinguished according to the level of homology,
the presence of conservative regions in the enzyme structure, and
structure of the active center. Each classification has its merits and
shortcomings, but none is exhaustive; therefore, they are often used
together. Data on beta-lactamase classification using division into
molecular classes and into functional groups are given in Table 2.

The first functional group includes enzymes of C molecular class (AmpC
type beta-lactamases) that can be encoded both by chromosomes and
plasmids [30]. A specific feature of this group is
their higher activity towards cephalosporins compared to penicillins,
and these enzymes also exhibit low sensitivity to inhibitors.

The second functional group, which is most comprehensive and diverse,
includes beta-lactamases of A and D classes. Genes of enzymes of this
group are incorporated into plasmids, and therefore efficiency of their
transfer among different strains and, as a result, their spreading
rates are very high. Group 2a includes beta-lactamases of Gram-positive
bacteria Staphylococcus spp. and Bacillus spp. These
enzymes are the most efficient against penicillins (with the exception
of oxacillin and its analogs). The best-known members of group 2b are
beta-lactamases TEM-1, TEM-2, and SHV-1. These enzymes also most
efficiently hydrolyze penicillins (except ureidopenicillins). Subgroup
2be includes so-called extended spectrum beta-lactamases (ESBL) that
are mutants of TEM-1 and SHV-1 enzymes and are able to cleave
efficiently both penicillins and cephalosporins of generations I-IV.
The same group includes numerous beta-lactamases of CTX-M type
efficiently hydrolyzing cefotaxime. A separate group 2br includes TEM
and SHV type inhibitor-resistant mutants. Group 2c includes PSE
beta-lactamases of Gram-negative bacteria able to hydrolyze
carbenicillin. Group 2d includes OXA type beta-lactamases that are
members of a separate molecular class D. Some of these enzymes have the
ESBL phenotype. They appear mainly in P. aeruginosa and A.
baumannii strains. It is assumed that mutation in position 167 is
the key one. Genes of group 2e cefalosporinases are localized on
chromosomes. They can be under control of both inducible (P.
vulgaris, C. diversus) and constitutive (Bacteroides
spp., Stenotrophomonas maltophilia) promoters. Group 2f is
formed by carbapenemases, i.e. by beta-lactamases able to hydrolyze
carbapenems.

The third functional group includes zinc-containing beta-lactamases.
Owing to this they are called metallo-beta-lactamases [31, 32]. According to molecular
classification, they belong to class B. Enzymes of this group
efficiently cleave all types of beta-lactam antibiotics including
carbapenems. They are resistant to inhibitors of clavulanic acid,
sulbactam, and tazobactam, but in culture they can be inhibited by
chelating agents like EDTA.

All beta-lactamases can be divided by mechanism of action into serine
(containing serine in the enzyme active center) and metalloenzymes.
Serine enzymes include beta-lactamases of molecular classes A, C, and
D, whereas class B beta-lactamases are metalloenzymes.

Most attention has been attracted recently to the problem of
plasmid-encoded ESBL [3, 33].
The term ESBL was initially used to designate TEM and SHV type
beta-lactamase mutants of 2be functional subgroup able to hydrolyze
oxyiminocephalosporins. Later the meaning of this term was
significantly extended. The following enzymes were included into the
ESBL group: enzymes with similar profile of substrate specificity but
structurally different from CTX-M and VEB type enzymes like TEM and SHV
type mutants with border activity towards cephalosporins such as
TEM-12; beta-lactamases with different resistance level, not included
into functional subgroup 2be, such as OXA type beta-lactamases and AmpC
beta-lactamase mutants with increased enzymic activity towards cefepime
[34].

Dissemination of Class A Extended Spectrum Beta-Lactamases

Dissemination of beta-lactamases, including ESBL, is actively studied in
many countries. The number of publications concerning ESBL abundance in
separate clinics, regions, and countries constantly increases. Data
obtained in Europe [35-37],
North [38] and South America [39], Canada [40], Asian region
countries [41, 42], and in
the Near East [43] are indicative of increased
resistance to beta-lactam antibiotics among pathogens of nosocomial and
outpatient infections, and of significance especially ESBL abundance,
which is a serious problem for public health. Comparative analysis of
these data indicates that the ESBL fraction among E. coli and
Kl. pneumoniae pathogens in countries of East and South Europe,
South America, and Asia has already reached 30% and more; in countries
of North Europe, North America, and Canada it is on average
significantly lower and does not exceed 10-15%. Although TEM and SHV
type beta-lactamases were mainly detected in Europe in the 1990s, the
situation changed sharply in the beginning of the XXI century and at
the present time the CTX-M type is prevalent in Europe, Asia, and South
America [44-49]. The increase
in the CTX-M type beta-lactamase fraction is exponential, and it is
already called pandemic [50]. It is supposed that
such high rate of spreading is due both to emergence of new mutants and
efficient gene transfer within plasmids and mobile genetic elements. In
the USA the situation is not changed, the SHV and TEM type
beta-lactamases are the most widespread, and the CTX-M type enzymes
were detected only in patients infected outside the country [35, 51].

Antibiotic resistance, including that to beta-lactam antibiotics, is
also investigated in Russia, and results have been published in a
number of works [52-55]. It
is noted that spreading of beta-lactam resistance among pathogens of
the Enterobacteriaceae family reaches on average 50-60% for
nosocomial infections, and it has already become a national problem.
Increase in the number of strains simultaneously containing genes of
two, three, and even four beta-lactamase types is found. The number of
such strains already reaches 30%. The ratio of ESBL types revealed in
Russia correlates with data on different ESBL type spreading in Eastern
Europe: the CTX-M beta-lactamases are significantly prevalent (about
80%), and beta-lactamases of SHV type are rather common. Enzymes of the
CTX-M-1 subcluster are the most often found CTX-M type genes. In recent
years significant increase in the CTX-M-9 subcluster beta-lactamase
frequency has been recorded [52, 56].

Variety of the Main Types of Class A Extended Spectrum
Beta-Lactamases

On the basis of available data on molecular mechanisms of bacterial
resistance to beta-lactam antibiotics and abundance of different
beta-lactamase molecular classes and functional groups, special
attention of researchers is given to investigation of three main ESBL
types: beta-lactamases of TEM, SHV, and CTX-M types. All contain serine
in the enzyme active center and have molecular mass about 29 kDa.
The main structural features of these enzymes will be briefly
considered below.

TEM type beta-lactamases. The first characterized enzyme of this
group was TEM-1 penicillinase detected in E. coli cells isolated
from blood of an infected patient [57]. Later
TEM-1-containing plasmids were found in strains of different members of
the Enterobacteriaceae family, in strains of Pseudomonas
aeruginosa, Haemophilus influenzae, Neisseria
gonorrhoeae, etc. Several years later the TEM-2 enzyme was
isolated, which differs by a single amino acid mutation Gln39Lys with
the same profile of substrate specificity [58].
However, already the next detected enzyme TEM-3 was characterized by a
second mutation Glu104Lys in addition to the already described mutation
in position 39. This enzyme was able to hydrolyze cephalosporins, and
thus it had a wider spectrum of substrates [58].
Now over 170 members of this type of enzymes have been described and
the beta-lactamase database (http://www.lahey.org/studies) is steadily growing.
The primary structure of TEM-1 includes 288 amino acid residues. All
described derivatives of TEM type beta-lactamases are variants of TEM-1
beta-lactamase and differ from original enzyme by single amino acid
substitutions (from one to seven). Mutations are found in 60 positions,
but the frequency of mutations in each position is very different.
Ninety TEM type enzymes are ESBL. Figure 1 shows
the distribution of known mutations in amino acid sequence of TEM-1
beta-lactamase, which is based on information available in the GenBank
international database. Most frequent are mutations in positions 21,
39, 69, 104, 164, 182, 238, 240, 244, 265, and 275. Among them
mutations in positions 104, 164, 238, and 240 are keys for extension of
substrate specificity. Introduction of substitutions Glu104Lys,
Arg164Ser(His), and Glu240Lys into TEM-1 results in change in total
charge of the protein globule. Mutation Gly238Ser results in appearance
of enzymes able to destroy with equal efficiency cefotaxime and
ceftazidime, while enzymes with mutation Arg164Ser are more active
towards ceftazidime and less efficient against cefotaxime [6]. There are mutations resulting in appearance of
inhibitor-resistant enzymes (IRT, inhibition resistance phenotype,
subgroup 2br) [60]. The following positions of
mutations define resistance to inhibitors: 69, 244, 275, and 276 [61]. Combination of the first and second type
mutations makes it possible to obtain TEM variants exhibiting both
types of resistance, ESBL + IRT. Six pairs of beta-lactamases
(TEM-1 and TEM-98, TEM-3 and TEM-14, TEM-10 and TEM-23, TEM-30 and
TEM-99, TEM-34 and TEM-97, TEM-63 and TEM-64) are structurally
identical. The use of random and directed mutagenesis [62] as well as of insertional mutagenesis [63] made it possible to obtain artificially
synthesized mutants with extended activity spectrum and to even
forecast the emergence of new subtypes of TEM beta-lactamases before
their clinical isolation.

Fig. 1. Positions and types of amino acid substitutions in TEM
type beta-lactamases described before January 2010.

SHV type beta-lactamases. Enzymes of this type emerged second
after TEM type beta lactamases. The peculiarity of this type of
beta-lactamases produced by Klebsiella pneumoniae is the
presence of SHV-1 beta-lactamase encoded by chromosomes [1]. In this case the SHV-1 enzyme, encoded by the gene
localized on plasmids, was widespread. The SHV-2 mutant differing by a
single mutation Gly238Ser was the first described ESBL [64].

The database http://www.lahey.org/studies/ contains information
about 126 mutants of SHV type beta-lactamases. They are derivatives of
SHV-1 enzyme and differ from the latter not only by the presence of
point mutations, but by deletions (in SHV-9 and SHV-10) or inserts (in
SHV-16) as well. Figure 2 shows the distribution of
mutations recently detected in amino acid sequence of SHV-1
beta-lactamase using information from the GenBank database. Mutations
are found in 79 positions; a deletion in position 54 in SHV-9 and an
insert 163DRWET167 in SHV-16 are also described. Depending on the
substitution type, like in the case of TEM enzymes, mutant SHV differ
by the profile of substrate specificity towards cephalosporins. Most
frequent are mutations in positions 35, 238, and 240 and they are
considered as the key ones for changes in the substrate specificity
profile. Beta-lactamase SHV-10 with IRT resistance phenotype is
described. It differs from ESBL SHV-9 by a single amino acid
substitution, Ser130Gly, but it is not an ESBL itself.

Fig. 2. Positions and types of amino acid substitutions in SHV
type beta-lactamases described before January 2010.

CTX-M type beta-lactamases. Beta-lactamases of CTX-M type
received their name because they more efficiently hydrolyze cefotaxime
compared to ceftazidime. The first enzyme of this type was isolated in
1989 from an E. coli strain [65]. Later it
was called CTX-M-1. Genes of this enzyme group have plasmid
localization. Over 90 enzymes of this type are known. Among the A class
ESBL, the CTX-M group of beta-lactamases is the most heterogeneous
concerning amino acid sequence and, respectively, the encoding gene
structures. The CTX-M type beta-lactamases are now divided into five
subclusters [66]. Each subcluster consists of the
main enzyme (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) and its
mutants differing by one to several mutations. Distribution of CTX-M
type enzymes by subclusters and mutations described for each subcluster
is shown in Fig. 3. The homology between CTX-M type
beta-lactamases and other types of A class enzymes is marginally
pronounced (below 40%) [67]. Much higher homology
(over 70%) is observed for the chromosome-encoded enzymes from
Klebsiella oxytoca, Citrobacter diversus, Proteus
vulgaris, and Serratia fonticola [68].
This suggests that the plasmid-encoded enzymes of CTX-M type originated
from enzymes whose genes are incorporated in chromosomes. The
difference between CTX-M type beta lactamases on one side and the TEM
and SHV type enzymes on the other side concerning their substrate
specificity is that all of CTX-M type lactamases hydrolyze
cephalosporins, i.e. they are ESBL. Differences between CTX-M type
enzymes concern changes in catalytic activity towards different
cephalosporins such as cefotaxime, ceftazidime, and cefepime. It was
shown that there are also key mutations for enzymes of this type, such
as those in positions 167 and 240, leading to changes in the substrate
specificity profile. It is noted that the CTX-M type beta-lactamases
are often revealed in infections in outpatients [19].

Fig. 3. Division of CTX-M type beta-lactamases into subclusters
and positions of amino acid substitutions in them described before
January 2010.

Problems of Laboratory
Diagnosis of ESBL

The difficulty of detection of ESBL in clinical
practice should be considered as their important feature. Standard
microbiological methods of determination of infectious pathogen nature
and resistance to antibiotics are based on phenotypic characteristic of
microbial pathogens. They are based on estimation of the antibiotic
minimal inhibitory concentration (MIC) necessary for inhibition of cell
growth in culture as tested by different methods using panels of
antibiotics and their combinations [69, 70]. According to the antibiotic MIC level, the
National Committee for Clinical Laboratory Standards (NCCLS) (USA)
established criteria of sensitivity of Gram-negative microorganisms to
the third generation cephalosporins only for E. coli and
Klebsiella spp. However, ESBL production is described in
practically all members of the Enterobacteriaceae family and in
a number of other Gram-negative microorganisms. These methods require
rather much time, and data concerning antibiotic sensitivity of certain
strains can also be interpreted ambiguously. Neither traditional
microbiological method based on estimation of microbial phenotype
provides for detection of ESBL in all strains. The “inoculum
effect” or a sharp increase in the antibiotic MIC upon increase
in the pathogen inoculation dose was found, i.e. the determined level
of sensitivity can depend on the cell concentration in culture.
Resistance determination is significantly complicated in the presence
of several microbial resistance determinants, and the number of such
cases constantly increases. For example, production by E. coli
strains of AmpC beta-lactamases (C class) concealed the presence of
ESBL, and they were not detected when E-test (5 samples of 7) or a
Phoenix automatic analyzer (3 samples of 7) was used [71]. Pseudo-positive results showing the presence of
ESBL in negative samples were also obtained in the same work, and most
such kind of results (9 false results for 19 samples) was obtained
using diffusion on discs. At best, traditional microbiological methods
of ESBL detection make it possible to estimate the fact of the presence
of an enzyme, but they cannot give information concerning the presence
of a certain enzyme. Therefore, it becomes obvious that the use of
phenotypic methods for testing sensitivity of bacterial pathogens is
not enough for proper understanding of the nature and properties of
infectious agent [72, 73].
The necessity of choosing adequate methods of diagnosis for correct
determination of resistance type is actively discussed [74]. This information can be obtained using
molecular-biological methods of gene analysis.

Analysis of the above-described data shows that study of the base
sequence of beta-lactamase-encoding genes is necessary for
determination of resistance of Gram-negative microorganisms to
beta-lactam antibiotics and for identification of gene types and
existing mutations. Point mutations in genes (single nucleotide
polymorphism (SNP)) are single nucleotide positions in genomic DNA for
which in a certain population there are different variants of sequences
(alleles) [75]. The detection of SNPs requires
either identification of complete base sequence of the gene or
structure of its fragments. All presently available
molecular-biological methods of base sequence analysis are based on
amplification of genes from biological material. Polymerase chain
reaction (PCR) is most often used for this. It is necessary to use
additional methods for determination of complete or partial structure
of DNA synthesized during the reaction. The “gold standard”
of gene molecular typing is sequencing or determination of primary
structure, i.e. determination of base sequence in the gene strand.
Sequencing can be used to identify both known and new gene variants.
The main disadvantage of this method is its labor consumption and
relatively high cost. The use of sequencing is difficult when it is
necessary to detect several genes in the same sample, especially when
these genes belong to the same genetic group where distinctions include
separate mutations.

Multiplex PCR

For simultaneous
amplification of several genes multiplex (multiprimer) PCR or the
reaction of co-amplification of several DNA templates in the same
reaction medium using several pairs of primers are used. Design of
primers and detection of probability of unspecific fragment
amplification are very important for increase in specificity of the
method. There are now multiplex PCR techniques for simultaneous
amplification of gene fragments of three main ESBL types (TEM, SHV,
CTX-M) [76]. The method can assign the enzyme gene
to a certain beta-lactamase type. More detailed gene differentiation by
multiplex PCR is possible at the subcluster level for CTX-M type ESBL,
but in this case analysis is carried out only for the same type enzymes
(in this case CTX-M) [77, 78]. The multiplex PCR technique becomes increasingly
important in combination with different methods providing more detailed
structural characteristics of the gene. Thus, the method of multiplex
PCR was elaborated for simultaneous amplification of CTX-M type
beta-lactamases, but owing to amplification of genes of different
subclusters in the form of equal size fragments, further investigation
of their structure is necessary for the assignment of gene type [79].

Real-Time Polymerase Chain
Reaction

Real-time PCR (RT-PCR) is an actively developing
technology. Different types of fluorescent dyes are used as labels. To
determine the result of the labeled oligonucleotide hybridization,
either donor fluorophore energy transfer to an acceptor with a
different emission spectrum or energy transfer to a fluorescence
quencher is used.

TaqMan technology is based on a method of destructible probes.
Oligonucleotide is modified by a fluorophore and fluorescence quencher.
In the absence of a target the fluorophore and quencher are drawn
together and fluorescence is inhibited. In the presence of a specific
template the probe is hybridized to an amplicon, which results in its
destruction and emergence of fluorescence due to 5′ exonuclease
activity of Taq polymerase (the ability to hydrolyze DNA sequence in
the 5′→3′ direction). The use of this approach was
described for identification of a subcluster of CTX-M type ESBL genes
[80]. The method is characterized by high rate and
good productivity compared to standard PCR techniques.

A different variant of RT-PCR is the method of adjacent probes based on
use of two oligonucleotide probes hybridized to template DNA in the
immediate vicinity of each other. One probe includes a donor
fluorophore and the other contains an acceptor fluorophore. Probe
hybridization with the template brings the fluorophores together and
energy transfer from donor to acceptor (fluorescence resonance energy
transfer) occurs. This approach became widely adapted to SNP detection.
Thus, its abilities were demonstrated in detection of key mutations in
the SHV, TEM, and CTX-M type ESBLs [81-84]. The method is limited by the possibility of
simultaneous detection of only a few SNPs due to the limited set of
fluorescent labels.

Methods for Analysis of Amplification Products Used for Gene
Identification

Restriction fragment length polymorphism (RFLP) method. This
method is used for detection of polymorphous DNA regions. To do this,
the amplified gene is cleaved by an appropriate restriction
endonuclease and changes in restriction sites are determined [85]. For each gene type its own set of restriction
products is obtained and analyzed by electrophoresis. The method
indicates the presence of variations in base sequence but requires
further analysis of fragments or comparison with a separation profile
of known gene variants. This approach was applied to identification of
SHV type beta-lactamases in Pseudomonas aeruginosa strains by
the presence of key mutations in positions 35, 238, and 240 [86]. The method is simple enough in practice and can
be used for screening a large number of samples; its limitation is the
possibility of detection of only mutations localized in restriction
sites as well as complications in revealing numerous mutations.

Method of analysis of conformational distinctions between
single-stranded DNA fragments. This method (SSCP, single strand
conformation polymorphism) was elaborated for SNP analysis and is based
on variations of single-stranded DNA conformation after denaturation.
This is demonstrated by changes in electrophoretic mobility. The SSCP
shows the presence of changes in nucleotide sequence, but it requires
further analysis of fragments or comparison with the separation profile
of known gene variants. The method was used for analysis of TEM and SHV
type ESBL [87, 88].

Method of time-of-flight mass spectrometry. An alternative to
existing methods for detection of polymorphous DNA regions is
differential sequencing by time-of-flight mass spectrometry. The
ionized DNA molecules are separated from the substrate using MALDI
(matrix-assisted laser desorption/ionization) technique, accelerated in
an electric field, and sent to a detector via a vacuum chamber. The
registered time of motion is inversely proportional to the speed of the
molecule, which in turn is in direct proportion to the mass-to-charge
ratio of the molecule. The mass-to-charge ratio is unique for each
molecule and is defined exclusively by its base sequence; therefore,
the sensitivity of the method is very high. The time spent for analysis
of each sample by this method is only a few seconds. The possibility of
use of this approach for rapid detection of TEM type ESBL by key
mutations in positions 104, 164, and 238 was shown [89]. The sensitivity of the method is high, and 50
bacterial cells in a sample are enough for determination. Determination
of nucleotide type in mutation position and, respectively, recognition
of genes with various substitutions is possible. The method is
characterized by high productivity and the possibility of simultaneous
analysis of several samples. Among clear advantages of the method is
the possibility to detect new mutations that have not been previously
described.

High performance liquid chromatography method. The method of high
performance liquid chromatography (HPLC) under denaturing conditions
was elaborated for analysis of the product formed during amplification,
and it was used for detection of CTX-M type ESBL [90]. Genes of CTX-M type beta-lactamases were
amplified using multiplex PCR [78], and in this
case the fragments of genes belonging to different subclusters differed
in size. Then amplified fragments were mixed with an equal amount of
DNA of control strains producing CTX-M type beta-lactamases that were
characterized by gene typing. Mixtures were quickly heated for
denaturation and then cooled at a certain rate for formation of homo-
and hetero-DNA duplexes. The duplexes were analyzed by HPLC in an
acetonitrile gradient. The temperature was optimized to make possible
distinguishing 13 CTX-M beta-lactamase gene variants by the shape of
chromatographic peaks. To achieve maximal sensitivity and specificity,
it was necessary to analyze duplexes for CTX-M-1 subcluster at two
temperatures, 64 and 65°C, for subclusters CTX-M-2 and 25/26 at
64°C, and for CTX-M-9 at 67°C. The method is characterized by
high productivity (100 samples in 6 h), and its cost is significantly
lower than that for sequencing. The authors of this work also suppose
that it will be possible to follow the emergence of new mutations by
appearance of anomalous chromatographic peaks. A limitation of the
method is that it determines only some mutations.

Pyrosequencing method. Pyrosequencing has been proposed for
analysis of mutations. The method is based on detection of
pyrophosphate cleavage during DNA synthesis. Pyrophosphate is the first
substrate in a cascade of enzymatic reactions resulting in emission of
light. The intensity of the emitted light is in proportion with the
number of incorporated nucleotides. Detection of emitted light
intensity in real time makes possible rapid identification of short
gene fragments up to 100 bp. Pyrosequencing was used for SNP detection
in genes of CTX-M type ESBL [91] and of GES type
ESBL [92]. Determination of genetic subcluster for
CTX-M type beta-lactamases was possible during analysis of 13 bp long
DNA fragment. Analysis of another 16 bp gene region identified separate
subtypes of CTX-M beta-lactamases. The method is characterized by the
very high rate of analysis; total determination time, including PCR, is
3 h.

Hybridization. Hybridization on insoluble supports proposed by E.
Southern [93] is used for specific detection of
individual DNA fragments. This method is often used after DNA
fragmentation like that using RFLP, after which electrophoretically
separated fragments are transferred onto porous membrane by
immunoblotting. Hybridization (complementary interaction of
polynucleotide strand under study with labeled oligonucleotide) is used
for identification. In this case the labeled oligonucleotide is the
“probe”. The label activity is determined in DNA duplexes
on the support after washing. Hybridization analysis was used for
detection of ESBL and AmpC beta-lactamases upon screening Salmonella
enterica and Pseudomonas aeruginosa strains [94, 95]. The use of
hybridization in amplified DNA detection significantly increases the
specificity of gene fragment determination. Depending on the type of
the label introduced into the oligonucleotide probe (fluorescent dyes,
haptens), different ways of detection of hybridization complex are used
(direct measurement of fluorescence, detection using specific antibody
conjugates with enzyme). Approaches developed for gene detection by
hybridization on solid phase served as a prototype for design of
hybridization analysis on microarrays.

Method of hybridization analysis on microarrays. The first report
concerning the technology of biological microarrays for investigation
of gene expression level appeared in 1995 [96].
During the past 10 years this technology has significantly developed
and gains more areas for its application. A DNA-microarray presents a
set of spots distributed in a strictly determined order on a glass or
polymer surface of a small size (1-10 cm2); each spot
contains single-stranded oligonucleotides with a particular base
sequence. The number of such spots and thus the number of different
base sequences can reach 1·106/cm2, their
length varying from 15 to 1000 nucleotides. The method of hybridization
analysis on microarrays is based on amplification of analyzed DNA
region with simultaneous introduction of label and following
hybridization of the analyzed base sequence on the microchip surface
with oligonucleotides or DNA sequences immobilized in a definite order
[97]. The difference between hybridization
analysis on microarrays and classical hybridization is that
oligonucleotides called probes are immobilized on the support and the
label is introduced into the analyzed DNA. The result of hybridization
of the labeled DNA sample with immobilized oligonucleotides (probes) is
determined by the label activity on the support using special
devices – high resolution scanners. Mainly passive
hybridization is used in DNA microarrays, i.e. the interaction of the
DNA target with the immobilized oligonucleotides is a random process
and depends on several factors such as the length of DNA probe,
chemical composition of labeled DNA target, hybridization temperature,
composition of hybridization mixture, and the type of introduced
label.

Different types of labels for hybridization analysis on microarrays are
used. Radioactive label, usually P33 incorporated into the
phosphate residue of the nucleotide introduced into the DNA strand
during its synthesis, was proposed for labeling of the analyzed DNA [93]. In this case each DNA molecule contains several
atoms of radioactive phosphorus, the maximal number of which can be
equal to the number of nucleotides. The presence of several labels
within the analyzed DNA increases sensitivity of the hybridization
assay. The use of isotope technology in microarrays makes it possible
to achieve high sensitivity, although there are some limitations. First
of all, they are connected with insufficiently high resolution of the
film used as the detector as well as with difficulty of work with
radioactive materials.

The fluorescent label is now the most widespread in hybridization
analysis on DNA microarrays. In this case, dyes directly joined to the
sequence of interest can be used as generators of the fluorescent
signal [98]. The fluorescent label is usually
introduced into DNA during PCR as a component of primers or fluorescent
dye conjugates with dCTP or dUTP [99]. Methods of
hybridization analysis using nanoparticles of gold with following
visual [100] or electrochemical detection [101] have been described.

So-called “indirect” labeling with biotin was proposed, and
biotin is revealed by a conjugate of streptavidin with fluorescent dye
[102] or with horseradish peroxidase [103] or alkaline phosphatase enzymes [104].

Hybridization analysis on microarrays has significant advantages over
traditional molecular-biological methods, which include the possibility
of multiparametric determination, i.e. determination of numerous
parameters (from tens to hundreds and even thousands of genes and
mutations in them) in a single sample due to carrying out numerous
parallel reactions on the microchip surface under identical
conditions.

The microchip technology is a principally new level of laboratory
investigations making possible hundreds and thousands of parallel
reactions. Examples of the application of this method for
identification and typing of different genes responsible for emergence
of antibiotic resistance among microorganisms will be considered in
separate sections.

METHODS OF HYBRIDIZATION ANALYSIS ON MICROARRAYS FOR
IDENTIFICATION OF SEVERAL ANTIBIOTIC RESISTANCE DETERMINANTS

Microarrays for Gene
Identification

Potentials of hybridization analysis on microarrays
providing for multiparametric gene determination were demonstrated upon
revealing genes responsible for development of antibiotic resistance,
including that to beta-lactam antibiotics.

A method was proposed for determination of nine beta-lactamase types
(TEM, SHV, PSE, OXA, FOX, MEN, SMY, OXY, and AmpC) on a glass microchip
with fluorescent detection [105]. Specific probes
of about 1000 bp were amplified by PCR using specific primers. The
studied DNA was amplified by multiplex PCR with introduction of
fluorescent label. Hybridization was carried out at 70°C for
2 h. The possibility of determination of resistance genes using
just a single bacterium as a PCR template was shown. Thus, it is
supposed that the sensitivity of the method makes the analysis possible
without preliminary culturing of clinical samples. The possibility of
detection of two type resistance genes (OXY and PSE) in the same sample
was shown experimentally.

A method was developed for determination of 23 resistance genes and 25
virulence genes in Salmonella spp. and E. coli strains
[106]. Sequences up to 400-800 bp long, obtained
by PCR, were immobilized on a chip of membrane support. Digoxygenin,
revealed by the anti-digoxygenin antibody conjugate with alkaline
phosphatase, was used as the DNA label. The enzyme on the support was
detected colorimetrically. Beta-lactamases of TEM, CMY, and PSE types
were revealed among different antibiotic resistance determinants.

A method was proposed for typing St. aureus spp., E. coli
spp., and Ps. aeruginosa spp. strains with simultaneous
identification of resistance determinants of these bacteria to several
groups of antibiotics [107]. About 120 specific
probes of 200-800 bp were amplified from recombinant plasmids and
applied onto a glass microchip. Probes for pathogen type identification
were tested along with probes homologous to regions of genes encoding
virulence factors and antibiotic resistance determinants in St.
aureus strains to oxacillin-methicillin (mecA), gentamycin
(aacA-aphD), erythromycin (ermA), and penicillin
(blaZ), and in E. coli strains to penicillin
(blaTEM-106) and aminoglycosides (aacC2). Blood
samples were cultivated on special media without further DNA
amplification. Total DNA labeled by several types of fluorescent dyes
was used for analysis. Hybridization time was 18 h at 60°C.
Although DNA isolated from blood of infected patient contains bacterial
and human DNA mixture, high specificity of identification was achieved.
There was 100% coincidence between results of identification on the
microchip and phenotyping data for St. aureus spp. resistant to
penicillin, oxacillin, erythromycin, and gentamycin. Among advantages
of the method the following can be mentioned: i) recombinant plasmids
were used for synthesis of the probe panel, which makes easy extension
of probe amount and composition; ii) the use of total DNA for analysis;
iii) the absence of an amplification stage of the analyzed DNA; iv)
preliminary pathogen typing is not necessary. From the point of view of
ESBL identification, the use of sufficiently long probes is a
disadvantage because it makes it impossible to use this approach for
detection of point mutations in genes.

Hybridization analysis on microarrays with colorimetric detection was
elaborated for identification of 90 antibiotic resistance genes in
Gram-positive microorganisms [108]. For
identification of 81 genes, two probes specific for each homologous
region of the studied genes were chosen, while for nine genes one probe
was chosen for each. Altogether, 137 oligonucleotides of 26-33 bp in
length were immobilized on a microchip, and specificity of gene
detection was studied for 125 probes. Biotin was used as the label. It
was introduced into the DNA during PCR and then was detected on the
microchip surface by incubation with streptavidin–peroxidase
conjugate and following detection of enzyme activity. The required
specificity of analysis was achieved upon hybridization for 1 h at
60°C.

In this work advantages of hybridization on microarrays, which exhibits
higher specificity than PCR, are discussed. The use of oligonucleotide
probes also has advantages over longer PCR products due both to higher
specificity and reduced time of analysis. The possibility of automating
different stages of the method undoubtedly is among its positive
characteristics: a microchip is placed into a special Array Tube
(technology of the Clondiag company), which makes significantly simpler
the performance of stages of hybridization, washing, and detection. The
impossibility of specific detection of point mutations due to rather
long oligonucleotide probes can be considered as a drawback of the
method.

The same technology was used in a microarray elaborated for 47 genes
responsible for development of resistance to five groups of antibiotics
(beta-lactams, aminoglycosides, trimethoprim, sulfonamides,
tetracycline, chloramphenicol, and quinolones) in Gram-negative
bacteria [109]. The microarray was tested using
50 clinical strains of E. coli and 37 clinical strains of
Salmonella spp. In 99% of the cases the results of resistance
determinant detection by the microarray technique coincided with those
of PCR carried out in the form of several multiplex reactions. In E.
coli strains many more resistance genes were found (13 resistance
determinants were revealed in over 30% of the samples) compared to
Salmonella spp. (seven resistance determinants were found). As
to beta-lactamase identification, the method only allowed determination
of beta-lactamase TEM and SHV gene type. Genes of CTX-M and OXA type
ESBL were revealed only at the subcluster level (CTX-M-1, CTX-M-2,
CTX-M-9; OXA-1, OXA-2, OXA-7, and OXA-9).

A microarray was designed for simultaneous determination of 65 genes
responsible for development of resistance to macrolides in several
types of Gram-negative and Gram-positive bacteria [110]. A panel of 100 probes of 40-60 bases in length
was used for gene typing. The probes were applied onto a glass
microchip. Genomic DNA was isolated from bacterial cultures,
fluorescent label was introduced into it, and it was hybridized on the
microchip. Good specificity of the method was demonstrated, and it was
possible to reveal for the first time the presence of one of the
msr(SA) genes responsible for the efflux system in the Gram-negative
microorganism B. fragilis.

Microarrays for SNP Determination

Hybridization analysis on microarrays allows both identification of
genes and recognition of single structural distinctions in the probe
and in the hybridized sequence, which is necessary for SNP
determination. For this aim the method of allele-specific hybridization
is most often used.

Allele-specific hybridization on a glass microarray was used to
determine fluoroquinolone-resistant E. coli strains [111]. Oligonucleotide structures were proposed for
nucleotide polymorphism identification in the gyrA gene,
encoding amino acid substitutions in positions 83 and 87.
Oligonucleotides were composed of 19 bases, and the detected mutation
was in the central position. A group of four oligonucleotides differing
in the base at central position was used for detection of one mutation.
Optimization of oligonucleotide structures provided high specificity of
determination: in all studied cases the result of complementary
hybridization exceeded that for non-complementary hybridization by
4-13-fold. In the work an attempt was made to design universal probes
using inosine, but this resulted in significant lowering of the
analytical signals.

The method of hybridization analysis on microarray was described for
detection of several types of ESBLs (TEM, SHV, CTX-M-3, CTX-M-9, MOX,
CMY, DHA, ACC, FOX, MIR-1, and ACT-1) and plasmid-encoded AmpC
beta-lactamase, including detection of six point mutations in the SHV
beta-lactamase gene [112]. Oligonucleotide probes
of 19-23 bases in length were immobilized on a glass microarray. Two
oligonucleotides with base sequence complementary to the wild type and
mutant enzymes were used for detection of point mutations. An
asymmetric two-step PCR technique was used for amplification. In the
first step a mixture of specific primers was used for amplification of
the studied gene fragments. The second step was carried out at a higher
temperature of annealing with universal fluorescence-labeled primers in
higher concentrations. Optimization of conditions provided mainly
single-stranded molecules, which made it possible to exclude the stage
of DNA denaturation before hybridization. Rather good correlation was
established between data of typing on the microarray and data of
phenotyping and sequencing.

For identification of nine resistance determinants and one mutation in
the grlA gene encoding the α subunit of DNA topoisomerase
in clinical sample of St. aureus, a microarray was designed on
the basis of glass-immobilized oligonucleotides [113]. The oligonucleotides were 17-24 bases long. A
set of four oligonucleotides was used for detection of point mutation.
A fluorescent label was introduced into the DNA after amplification.
Hybridization on the microarray was carried out for 4 h at
42°C. The microarray enables simultaneous testing of bacterial
resistance to beta-lactams, tetracyclines, gentamycin, macrolides,
streptogramin A compounds, and fluoroquinolones. The detection limit
was 100 pg DNA, which corresponds to 104 bacterial cells.

An original technology of gel biological chips for gene and SNP
identification was designed at the Engelhardt Institute of Molecular
Biology [114]. In this method oligonucleotide
probes are immobilized on the microarray surface in the form of a
hemispherical hydrogel of cells from 100 µm to 1 mm in
diameter. Immobilization is carried out with formation of covalent
bonds upon ultraviolet irradiation. The high-porosity gel makes
possible immobilization of oligonucleotides of up to 200 bases. A
fluorescent dye is introduced into the analyzed DNA. Electric fields
transverse to the microarray surface introduces hydrodynamic flows that
accelerate the kinetics of the labeled DNA hybridization with
oligonucleotides. The probe concentration in the gel microarrays
exceeds almost 103-fold that in the case of planar
microarrays, which significantly increases the sensitivity of the
method. This approach was used for detection of antibiotic-resistant
forms of Mycobacterium tuberculosis [115].
For this aim SNPs that define resistance to rifampicin were determined
in 28 positions of the rpoB gene as well as SNPs responsible for
resistance to isoniazid were determined in 11 positions of the
katG gene, in five positions of the promoter of the inhA
gene, and in five positions of the regulatory region of gene
ahpC-oxyR. Fluorescent label was introduced into PCR primers,
and the analysis time was 12 h.

The variety of beta-lactamase types and variants makes rather
complicated the problem of their identification for clinical laboratory
diagnosis and epidemiological monitoring of the emergence and spreading
of infection. The existence of several molecular classes of enzymes as
well as their variability makes it necessary to use methods able to
provide for simultaneous detection both of several gene types and
mutations in them. At first sight it may seem that the problem of ESBL
identification is more highly specialized, because formally ESBL are a
small part of the beta-lactamase family and belong to one functional
subgroup of 12 already described. However this subgroup is one of most
numerous concerning the detected and described gene variants and point
mutations in them. Therefore ESBL gene typing, i.e. determination of
genotype of an enzyme produced by the bacterial strain under study,
suggests simultaneous determination of several gene types and several
hundreds of mutations in them. Data on the use of various
molecular-genetic methods for detection of the main ESBL types that are
based on PCR modifications and analysis of amplification products by
different methods are given in Table 3. The use
of methods based on PCR and the combination of PCR with methods of
product analysis based on gel electrophoresis, chromatography, and
mini-sequencing usually allows determination of only a limited number
of genes and SNPs in a single analysis. Therefore, these methods cannot
be used when identification of enzyme genotype is required. Among the
total variety of described molecular methods, methods of
multiparametric hybridization analysis on microarrays, considered in
this section, seem to be the only adequate technologies for ESBL
multiparametric gene typing.

The method of hybridization analysis with biotin label revealed by the
streptavidin–peroxidase conjugate with subsequent
chemiluminescent enzyme detection was proposed for identification of
the most widespread variants of CTX-M type beta-lactamase genes [119]. Biotin was introduced into reverse primers
used for multiplex PCR. Primer structure was selected so that amplified
fragments of the CTX-M beta-lactamase gene belonging to four different
genetic subclusters differed in size. Enzymes of CTX-M-8 and CTX-M-25
types were combined in a single subcluster. On the membrane macroarray
oligonucleotides were immobilized as bands in an immunoblotter. For
hybridization, the array was turned through 90° relative to the
initial orientation, and the immunoblotter channels were filled with
the amplified DNA solutions in hybridization buffer. The peculiarity of
this method is hybridization performed in kinetic regime; to increase
specificity of analysis, the sample was washed at enhanced temperature.
After washing, the amount of label in DNA duplexes was determined on
the support by peroxidase activity in the chemiluminescence
reaction.

Differences in Tm of selected oligonucleotide panels did not
allow selection of the same conditions for hybridization analysis for
all four genetic subclusters. Because of this, a separate membrane
array and different temperatures of washing buffer were used after
hybridization (from 58 to 65°C) for detection of mutations within
each subcluster. To determine which array is necessary for typing of
any sample, the subcluster type was determined by the size of amplicon
obtained in multiplex PCR. The selected oligonucleotides enabled
identification of the presently most widespread gene variants of the
CTX-M type beta-lactamases: CTX-M-1, -2, -3, -9, -14, and -15. High
productivity of this method (43 samples can be tested in parallel),
rather short time of analysis (about 7 h), and inexpensive equipment
can be considered as its advantages. Limitations of this method are
both unusual conditions of hybridization analysis for different
subclusters of CTX-M beta-lactamase genes and a limited number of
determined SNPs (four of each SNP for subclusters CTX-M-1, CTX-M-2, and
CTX-M-8 and five for subcluster CTX-M-9).

The method of hybridization analysis on glass microarrays was proposed
for TEM type beta-lactamase gene typing on the basis of determination
of all SNPs [120]. The fluorescent label used in
this method was introduced into the gene during PCR in the form of
modified deoxyribocytosine triphosphates (dCTP-Cy5). The scheme of the
method of hybridization analysis with fluorescent detection is shown in
Fig. 4a. Amplified labeled DNA was fragmented
before hybridization by DNase for obtaining approximately 50-base-long
oligonucleotide strands. It was shown earlier that this increases
hybridization efficiency [121]. Hybridization was
followed by scanning of fluorescence activity on the microarray
surface. For one SNP determination a set of four oligonucleotides with
unique base sequence corresponding to the TEM beta-lactamase gene
structure in a given region and differing from each other by just a
single nucleotide in the central position, which could be one of four
nucleotides A, G, C, or T, was used (Fig. 5a). The
most stable duplex is formed in the case of the labeled fragment
hybridization with a fully complementary oligonucleotide, and
correspondingly, in this case a higher analytical signal (PM, perfect
match) is detected (Fig. 5b). If duplexes are
formed during hybridization with three other oligonucleotides or with
only some of them, then the observed signal (MM, mismatch) is
indicative of the level of unspecific hybridization.

Fig. 5. a) Scheme of point mutation detection in gene by
allele-specific hybridization. b) The result of point mutation
detection in a gene by hybridization analysis on microchip. c)
Representation of results of hybridization analysis for detection of a
single mutation in a gene in relative units.

Molecular design of oligonucleotide structures for 41 SNPs in TEM
beta-lactamase genes was carried out in this work. The following rules
of oligonucleotide structure selection for SNP recognition were
observed [122]: (i) oligonucleotides should be
17-26 bases long; (ii) the G + C content should be in the range of
35-70%; (iii) the probability of dimer and cyclic structure formation
should be minimal; (iv) Tm differences for different
oligonucleotide groups should not exceed 5-10°C.

The principle of internal signal normalization was used for nucleotide
type determination in the analyzed gene position. For this aim all
results of hybridization within the set of four oligonucleotides were
normalized to the signal of complementary hybridization. In this way
hybridization signals were obtained in relative units: signal of
complementary hybridization became equal to 1, whereas the other three
signals of non-complementary hybridization were expressed in its parts
(Fig. 5c):

RIPM =
IPM/IPM = 1,

RIMM =
IMM/IPM,

where IPM is intensity of signal of hybridization
with fully complementary oligonucleotide, IMM is
intensity of signal of hybridization with the other three
oligonucleotides, RIPM is fraction of
complementary hybridization, RIMM is fraction of
noncomplementary (unspecific) hybridization.

Parameter of unspecific hybridization fraction
RIMM =
IMM/IPM was used for estimation of
SNP detection specificity. If the fraction of unspecific hybridization
exceeded 0.7, then the SNP detection was considered as unspecific.
Molecular design of oligonucleotide structures for SNP detection in TEM
type beta-lactamases allowed highly specific detection of mutations:
over 99% of RIMM values did not exceed 0.4, and only
a single group of oligonucleotides was characterized by
RIMM = 0.52. The authors of this work tried to
reduce significantly the hybridization time from three hours to
15 min in order to elaborate a method for express analysis of
resistant strains that could be used in clinical laboratories. It was
found that in the case of 1-h-long hybridization reaction, in 84% of
cases RIMM values are not higher than 0.4, 14% of
RIMM values are not higher than 0.6, while in the
case of a single mutation an RIMM value close to 0.7
is observed. Reduction of hybridization time to 30 min revealed
similar specificity with lower reproducibility. Further reduction of
hybridization time to 15 min resulted in significant lowering of
the detection specificity: six positions were identified incorrectly,
while 21% of RIMM values exceeded 0.7. Thus, it was
found that hybridization time could be reduced to 30 min. In this
case total time of analysis was 3.5 h, which is a significant
improvement compared to standard phenotypic tests taking 2-3 days.

We have used the same approach for elaboration of hybridization analysis
on microarrays for gene typing of CTX-M type beta lactamases [123] that differ from other class A ESBL by lower
homology between the same type genes. Because of this, oligonucleotide
structures for genetic subcluster identification were also selected
along with oligonucleotides for identification of point mutations in
genes.

On the whole, the CTX-M type beta lactamases differ from TEM and SHV
type beta-lactamases by higher G–C content, which resulted in a
higher level of unspecific hybridization compared to previous data [120]. Thorough molecular design of specific
oligonucleotide structures was carried out for lowering the level of
unspecific hybridization. Introduction of artificial substitutions into
oligonucleotide structure was used to increase specificity of the
central oligonucleotide detection. Results of testing of the CTX-M-3
beta-lactamase-producing control strain by hybridization analysis on
microarrays using fluorescent dye Cy3 as DNA label are shown in Fig. 6a. Specificity of separate SNP detection during
hybridization at 45°C was not high enough: 16% of
RIMM values did not exceed 0.7. On increase of
hybridization temperature to 47°C the specificity of SNP
identification was improved insignificantly: the fraction of
RIMM values below 0.4 increased, but 10% of
RIMM values exceeded 0.7 (Fig. 6b).

Since hybridization efficiency and specificity are defined by the
oligonucleotide strand secondary structure that can be influenced by
the introduced label, we studied the effect of an alternative biotin
label having significantly smaller size compared to fluorescent labels
of the cyanin group [124]. The scheme of
hybridization analysis with biotin label is shown in Fig. 4b. Biotin was introduced into the beta-lactamase gene
during PCR (in the form of dUTP-biotin). The introduced biotin was
revealed using the streptavidin–horseradish peroxidase (HRP)
conjugate and HRP colorimetric detection with substrates whose
oxidation is accompanied by formation of intensely stained product
adsorbed on the glass surface near the enzyme molecules. Figure 6c shows results of testing of the CTX-M-3
beta-lactamase-producing control strain by hybridization analysis on
microarrays using biotin label. The specificity of SNP revealed by
hybridization analysis with biotin label was significantly higher even
at 45°C. Data in Fig. 7 compare specificity in
revealing 19 different SNPs described for the CTX-M-1 subcluster
beta-lactamases upon hybridization analysis with fluorescent and
colorimetric detection. The use of biotin label provided the best
specificity in detection of point mutations: 94% of
RIMM values were below 0.4. All in all,
oligonucleotide probe structures were selected for detection of 67 SNPs
in CTX-M type beta-lactamase genes. The elaborated method was proven
using a collection of 94 clinical microbial strains of the
Enterobacteriaceae family. A full 100% coincidence was obtained
between gene typing by hybridization analysis on the microarray and
complete gene sequencing.

Thus experiments on multiparametric SNP typing in TEM and CTX-M type
beta-lactamase genes by allele-specific hybridization on microarrays
revealed the good potential of this method. The peculiarity of the
considered approach is its sufficient universality and the possibility
of extension of the type of identified genes and mutations.

Later this was confirmed during elaboration of integrated technique for
simultaneous gene typing for the three most clinically important ESBL
(TEM, SHV, and CTX-M types) on the basis of SNP detection in encoding
genes [125]. Two multiplex PCRs were elaborated
for gene amplification: in one of them genes of TEM and SHV type
beta-lactamases were amplified, while the other was used for
amplification of the CTX-M type beta-lactamase two gene fragments
belonging to all four subclusters and comprising in aggregate the
full-size gene (open reading frame). The fluorescent label Cy3
introduced into the gene during PCR was used as the DNA label. A set of
four oligonucleotides differing in nucleotide type in the central
position or in that close to central was used for one SNP detection. It
was proposed to place oligonucleotide groups on the microchip in the
form of modules, each of which contains oligonucleotide groups for
detection of mutations in the same type beta-lactamase genes. This will
make possible in future to add new oligonucleotide groups for revealing
new mutations in analyzed gene types as well as new oligonucleotide
modules for revealing new beta-lactamase types.

The main problem of integration of the earlier developed oligonucleotide
panels for gene typing of TEM, SHV, and CTX-M type beta-lactamases into
a single microarray is how to optimize the conditions providing the
efficient hybridization of structurally different oligonucleotides with
retention of high sensitivity and specificity of analysis. Due to
structural differences between genes of studied ESBL types and higher
G–C content in CTX-M type genes, hybridization temperature was
increased to 47°C instead of that chosen earlier for gene typing of
TEM type beta-lactamases to increase the specificity of the analysis.
Hybridization was carried out for 1 h under automatic conditions
in a hybridization tube. Artificial substitutions were introduced into
some oligonucleotide sequences to lower the probability of formation of
dimer and other stable secondary structure elements. Molecular design
of specific oligonucleotide structures provided very high specificity
of SNP determination: in 94% of oligonucleotides the fraction of
unspecific hybridization was below 0.4. The developed method allows
simultaneous detection of over 150 SNP, which is much higher than the
number of parameters simultaneously determined by other methods.

The integrated microchip was tested using 60 samples already
characterized by standard phenotyping tests for strain sensitivity to
panels of antibiotics and their combinations with inhibitors.
Hybridization analysis on the microarray revealed 93% sensitivity and
100% specificity of this method. ESBLs of one or several types were
found in 54 samples. For four samples phenotypically identified
resistance could not be explained by ESBL production. Successful
identification of different gene type mixtures (like TEM-1 and SHV-12;
TEM-1, SHV-1, and CTX-M-15) and mixture of two genes belonging to the
same gene type (like CTX-M-15 and CTX-M-14b; SHV-1 and SHV-14) can be
considered as advantages of the developed method. These results were
confirmed by sequencing. It should be noted that detection of a mixture
of two genes on a microarray is more clear and reliable, especially for
genes of the same type, whereas in the case of sequencing detection of
two chromatographic peaks causes difficulties especially for automatic
data processing.

The variety of beta-lactamases and extremely rapid spreading of known
resistance determinants and emergence of new determinants and new
combinations of previously described resistance determinants make
necessary elaboration of adequate methods for their clinical
diagnostics for choosing the correct course of therapy and monitoring
of spreading of infectious diseases. Molecular-biological methods of
gene structure analysis are indispensable for solution of these
problems. Amplification and hybridization technologies have already
provided a qualitatively new level of diagnosis of many infectious and
genetic diseases.

Antibiotic resistance of infectious pathogens was tested at the
“micro” level when resistance is determined in a single
clinic or in a separate group of patients, as well as at
“macro” level in inter-center, national, and international
studies when hundreds, thousands, and tens of thousands of pathogen
strains collected in different clinics are analyzed. For large-scale
investigations it is desirable to have methods allowing detection of
numerous parameters in the same reaction that are characterized by
sufficient sensitivity and specificity along with high productivity.

Broad introduction of sequencing for these aims is difficult due to
complication and laboriousness of the method, the necessity of
specialized expensive equipment, and to rather high cost of analysis.
Multiparametric analysis on microarrays seems to be a convenient
alternative to sequencing. Multiparametric determination of genes and
mutations in them by hybridization analysis on microarrays has great
potential for use in investigation of molecular mechanisms of infection
resistance and spreading.

This work was supported by Russian Federal Program “National
Technological Resources 2007-2011” (State contract
GP/07/442/NTB/K) and a grant of the German Federal Ministry of
Education and Science within the framework of the GenoMik (Genome
Research on Microorganisms) project.